In addition to environmental problems such as global warming and air pollution, from concerns related to the energy supply for transport such as the significant increase in crude oil prices and crude oil depletion expected in the near future (peak oil), in recent years, development of alternative energy to petroleum is a very important issue. Cellulose-based biomass, such as plant biomass and lignocellulose, which is the most abundant renewable energy source on the earth, is expected as an alternative resource to petroleum.
By culturing an Aspergillus fungus (koji mold) producing a saccharifying enzyme on the surface of the solid biomass such as rice straw and corn stover, it is possible to subject the biomass to a saccharification treatment. By using a transformant obtained by introducing a gene for a saccharifying enzyme with higher saccharification capability into an Aspergillus strain, it is possible to improve the efficiency of the saccharification treatment.
On the other hand, when introducing a foreign gene into a microorganism such as an Aspergillus strain for transformation, in order to selectively pick only microorganisms into which the foreign gene of interest has been introduced, a method of using an auxotrophic strain as a host which is deficient of the pyrG gene (orotidine-5′-phosphate decarboxylase), sC gene, niaD gene and the like has been generally used (see, for example, Non-Patent Document 1 or 2). For example, when using a strain that became auxotrophic for uridine due to deletion of the pyrG gene as a host strain and culturing in a uridine-free medium after introducing thereinto a combination of the gene of interest and the pyrG gene, since only transformants are able to grow, it is possible to efficiently select genetically modified fungi.
In addition, a method has been known to date for improving the homologous recombination efficiency due to deletion of the ligD gene (encoding a DNA ligase) (for example, see Non-Patent Document 3) and also for recycling the pyrG gene by utilizing homologous recombination (marker recycling) (for example, see Non-Patent Document 4). When carrying out gene recombination, by deleting the ligD gene, it is possible to suppress random introduction of the target gene into the genome and to efficiently construct a strain in which the gene is introduced into a targeted site. In addition, through the marker recycling method, it is possible to use one marker gene for the selection in multiple gene transfers, although only the selection for a single gene transfer has been carried out conventionally for one marker gene.